Summary Proteomics is essential for deciphering how biomolecules interact as a system and for understanding the functions of cellular systems in human diseases. However, the unique characteristics of the human proteome, which include the large dynamic range of protein expression and the extreme complexity resulting from a plethora of post-translational modifications (PTMs) and sequence variations, make such analyses challenging. A comprehensive analysis of all proteoforms in the human proteome that arise from genetic variations, alternative splicing, and PTMs, is essential for gaining a transformative understanding of disease mechanisms and identifying new therapeutic targets. The emerging top-down mass spectrometry (MS)-based proteomics, which is based on analysis of intact proteins, is arguably the most powerful method to comprehensively characterize proteoforms to decipher the PTM codes together with genetic variations. However, top-down MS-based proteomics still faces significant challenges in terms of protein solubility, protein separation, and detection of low- abundance proteins. In this multiple-PI project, we will develop novel approaches enabled by nanotechnology and materials chemistry to overcome the challenges facing top-down proteomics in a comprehensive manner. The specific objectives of this proposal are: 1) To develop novel top-down MS-compatible surfactants that can effectively solubilize all categories of proteins including membrane proteins. 2) To develop novel chromatography materials based on mesoporous silica nanomaterials with uniform pore sizes and new strategies for high- resolution multi-dimensional liquid chromatography for effective separation of intact proteins. 3). To develop functionalized multivalent nanoparticles for highly specific enrichment of low-abundance proteins and proteins with PTMs. Our highly interdisciplinary approach is a perfect marriage between materials chemistry/nanotechnology and top-down MS-based proteomics. It is built on an existing collaboration between the two PIs that has led to significant preliminary results. We envision the development of the proposed technologies will significantly advance the burgeoning field of top-down proteomics. The success in our research will provide innovative tools for gaining transformative insights into the molecular basis of diseases and developing new strategies for the early diagnosis, prevention, and better treatment of human diseases.